This project will continue to develop new techniques and machinery for rapidly freezing skeletal muscles as they are stimulated and monitored electrophysiologically. Neuromuscular junctions frozen at the precise moment of neurotransmission will be prepared by the methods of freeze fracture and freeze substitution for examination in the electron microscope. In this way, the fleeting structural changes that occur during secretion of acetylcholine from the nerve and its reception by the muscle will be revealed in their natural form. This structural data will help to elucidate the molecular mechanisms that underlie normal neuromuscular transmission, and will form a basis of comparison with neuromuscular pathology. Work will aso continue on the new quick-freeze, freeze-dry technique which promises to elucidate the detailed interrelationships between membrane molecules and cytoplasmic filaments in a variety of cellular processes, including hormone uptake, organelle translocation, and nonmuscle cell motility.